Mammalian cytokine-like factor 7

ABSTRACT

Novel mammalian zcyto7 polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods including antibodies and anti-idiotypic antibodies.

[0001] This application is a continuation of U.S. application Ser. No.09/633,452, filed Aug. 7, 2000, which is a continuation of U.S.application Ser. No. 09/066,745, filed Apr. 24, 1998, which claims thebenefit of U.S. Provisional Application Serial No. 60/071,676 filed Jan.16, 1998; and U.S. Provisional Application Serial No. 60/044,886 filedApr. 25, 1997.

BACKGROUND OF THE INVENTION

[0002] Proliferation and differentiation of cells of multicellularorganisms are controlled by hormones and polypeptide growth factors.These diffusable molecules allow cells to communicate with each otherand act in concert to form cells and organs, and to repair andregenerate damaged tissue. Examples of hormones and growth factorsinclude the steroid hormones (e.g. estrogen, testosterone), parathyroidhormone, follicle stimulating hormone, the interleukins, plateletderived growth factor (PDGF), epidermal growth factor (EGF),granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO) and calcitonin.

[0003] Hormones and growth factors influence cellular metabolism bybinding to proteins. Proteins may be integral membrane proteins that arelinked to signaling pathways within the cell, such as second messengersystems. Other classes of proteins are soluble molecules, such as thetranscription factors.

[0004] Of particular interest are cytokines, molecules that promote theproliferation and/or differentiation of cells. Examples of cytokinesinclude erythropoietin (EPO), which stimulates the development of redblood cells; thrombopoietin (TPO), which stimulates development of cellsof the megakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia or receiving chemotherapy for cancer. The demonstrated invivo activities of these cytokines illustrates the enormous clinicalpotential of, and need for, other cytokines, cytokine agonists, andcytokine antagonists.

SUMMARY OF THE INVENTION

[0005] The present invention addresses this need by providing a novelpolypeptide called cytokine-like factor 7,hereinafter referred to asZcyto7 and related compositions and methods.

[0006] Thus, one aspect of the present invention provides for anisolated Zcyto7 polypeptide having amino acid sequences as follows. Boththe human and the mouse cDNAs have been discovered. The human sequencesare defined by SEQ ID NOs: 1 and 2. The murine nucleotide and amino acidsequences are defined by SEQ ID NOs: 11 and 12.

[0007] The nucleotide sequence of SEQ ID NO:1 contains an open readingframe encoding a polypeptide of about 180 amino acids with the initialMet as shown in SEQ ID NO:1 and SEQ ID NO:2. A predicted signal sequenceis comprised of amino acid residues 1-20, and the resultant predictedmature Zcyto7 polypeptide is represented by the amino acid sequenceextending from amino acid residue 21, a glutamine to and including aminoacid residue 180 a phenylalanine, also represented by SEQ ID NO: 14.Peptide mapping data indicate that mature Zcyto7 can be comprised of anumber of N-terminal mature variants including the amino acid sequenceextending from amino acid residue 23, an arginine to and including aminoacid residue 180 of SEQ ID NO: 2, also defined by SEQ ID NO: 36; aminoacid sequence extending from amino acid residue 27, a serine to andincluding amino acid residue 180 of SEQ ID NO: 2, also defined by SEQ IDNO: 37; the amino acid sequence defined by amino acid residue 30, alysine, to and including amino acid residue 180 of SEQ ID NO: 2, alsodefined by SEQ ID NO: 38; amino acid sequence extending from amino acid28, a lysine, to and including amino acid residue 180 of SEQ ID NO:2,also defined by SEQ ID NO: 41 and the amino acid sequence extending fromamino acid residue 53, a methionine, to and including amino acid residue180, also defined by SEQ ID NO: 42. The only observed cleavage at thecarboxyl terminus is the phenylalanine at position 180 can be cleavedoff. This can occur in all of the above-defined mature Zcyto7polypeptides an example of which is shown by SEQ ID NO:43. Additionalvariants of human Zcyto7 are defined by SEQ ID NOs: 15-25. Within anadditional embodiment, the polypeptide further comprises an affinitytag.

[0008] SEQ ID NOs: 11 and 12 define murine Zcyto7 wherein the matureprotein extends from amino acid residues amino acid residue 21, ahistidine, to and including amino acid residue 180 a phenylalanine, alsodefined by SEQ ID NO: 39; or as an alternative splice site from aminoacid residue 23, an arginine, to and including amino acid 180 alsodefined by SEQ ID NO: 40. The present invention is also comprised ofpolypeptides having an amino acid sequence at least 90% identical, morepreferably 95%, 97% or 99% identical to those Zcyto7 polypeptidesdefined in above.

[0009] An additional embodiment of the present invention relates to apeptide or polypeptide which has the amino acid sequence of anepitope-bearing portion of a Zcyto7 polypeptide having an amino acidsequence described above. Peptides or polypeptides having the amino acidsequence of an epitope-bearing portion of a Zcyto7 polypeptide of thepresent invention include portions of such polypeptides with at leastnine, preferably at least 15 and more preferably at least 30 to 50 aminoacids, although epitope-bearing polypeptides of any length up to andincluding the entire amino acid sequence of a polypeptide of the presentinvention described above are also included in the present invention.Examples of said polypeptides are defined by the amino acid sequences ofSEQ ID NOs: 25-35. Also claimed are any of these polypeptides that arefused to another polypeptide or carrier molecule.

[0010] The present invention further comprises a polypeptide defined bySEQ ID NOs: 15-25 wherein the amino termini of said polypeptides aremodified and begin at either amino acid residue 3, an arginine; aminoacid residue 7, a serine; amino acid residue 8, a lysine; amino acidresidue 10, a lysine or amino acid residue 33 methionine.

[0011] The present invention is further comprised of a polypeptidewherein the polypeptide is a polypeptide defined by SEQ ID NOs: 2, 12,14-25 and 36 to 42 wherein the amino acid sequences end at theisoleucines at amino acid residue 179 of SEQ ID NO: 2, at amino acidresidue 159 of SEQ ID NOs: 14-25, which corresponds to amino acidresidue 157 of SEQ ID NO:36, amino acid residue 153 of SEQ ID NO:37,amino acid residue 150 of SEQ ID NO:38, amino acid residue 159 of SEQ IDNO: 39, amino acid residue 157 of SEQ ID NO:40, amino acid residue 152SEQ ID NO:42, amino acid residue 127 of SEQ ID NO:42.

[0012] The present invention is further comprised of an isolated peptideor polypeptide of the above-described peptides or polypeptide having anamino acid sequence modified by addition, deletion and/or replacement ofone or more amino acid residues and which maintains the biologicalactivity of said peptide or polypeptide.

[0013] Within a further aspect of the invention there is provided achimeric polypeptide consisting essentially of a first portion and asecond portion joined by a peptide bond. The first portion of thechimeric polypeptide consists essentially of (a) a Zcyto7 polypeptide asdescribed above (b) allelic variants of the polypeptides describedabove. The second portion of the chimeric polypeptide consistsessentially of another polypeptide such as an affinity tag. Within oneembodiment the affinity tag is an immunoglobulin F_(c) polypeptide. Theinvention also provides expression vectors encoding the chimericpolypeptides and host cells transfected to produce the chimericpolypeptides.

[0014] Another aspect of the present invention provides for isolatednucleic acid molecules comprising a polynucleotide selected from thegroup consisting of: (a) a nucleotide sequence encoding the Zcyto7polypeptides described above; (b)a nucleotide sequence encoding thepolypeptides of SEQ ID NOs: 14-40 and (c) a nucleotide sequencecomplementary to any of to any of the nucleotide sequences in (a) or(b).

[0015] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably 95%, 97%, 98%, or99% identical to any of the nucleotide sequences in (a), (b) or (c)above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence of (a) (b) or (c) above. An additional nucleic acid embodimentof the present invention relates to an isolated nucleic acid moleculecomprising an amino acid of an epitope-bearing portion of a Zcyto7polypeptide.

[0016] Within another aspect of the invention there is provided anexpression vector comprising (a) a transcription promoter; (b) a DNAsegment encoding a polypeptide described above, and (c) a transcriptionterminator, wherein the promoter, DNA segment, and terminator areoperably linked.

[0017] Within a third aspect of the invention there is provided acultured eukaryotic cell into which has been introduced an expressionvector as disclosed above, wherein said cell expresses a proteinpolypeptide encoded by the DNA segment.

[0018] In another embodiment of the present invention is an isolatedantibody that binds specifically to a Zcyto7 polypeptide describedabove. Also claimed is a method for producing antibodies which bind to aZcyto7 polypeptide comprising inoculating a mammal with a Zcyto7polypeptide or Zcyto7 epitope-bearing polypeptide so that the mammalproduces antibodies to the polypeptide; and isolating said antibodies.

[0019] These and other aspects of the invention will become evident uponreference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The teachings of all of the references cited herein areincorporated in their entirety herein by reference.

[0021] The term “affinity tag” is used herein to denote a polypeptidesegment that can be attached to a second polypeptide to provide forpurification or detection of the second polypeptide or provide sites forattachment of the second polypeptide to a substrate. In principal, anypeptide or protein for which an antibody or other specific binding agentis available can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A [Nilsson et al, EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)], glutathione Stransferase [Smith and Johnson, Gene 67:31 (1988)], Glu-Glu affinity tag[Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985)],substance P, FLAG™ peptide (Hopp et al., Biotechnology 6:1204-10 (1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107 (1991). DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

[0022] The term “allelic variant” denotes any of two or more alternativeforms of a gene occupying the same chromosomal locus. Allelic variationarises naturally through mutation, and may result in phenotypicpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequence. The term allelic variant is also used herein todenote a protein encoded by an allelic variant of a gene.

[0023] The term “expression vector” denotes a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments may include promoter andterminator sequences, and may optionally include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or may contain elements of both.

[0024] The term “isolated”, when applied to a polynucleotide molecule,denotes that the polynucleotide has been removed from its naturalgenetic milieu and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, but mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art. See for example, Dynanand Tijan, Nature 316:774-78 (1985). When applied to a protein, the term“isolated” indicates that the protein is found in a condition other thanits native environment, such as apart from blood and animal tissue. In apreferred form, the isolated protein is substantially free of otherproteins, particularly other proteins of animal origin. It is preferredto provide the protein in a highly purified form, i.e., greater than 95%pure, more preferably greater than 99% pure.

[0025] The term “operably linked”, when referring to DNA segments,denotes that the segments are arranged so that they function in concertfor their intended purposes, e.g. transcription initiates in thepromoter and proceeds through the coding segment to the terminator

[0026] The term “polynucleotide” denotes a single- or double-strandedpolymer of deoxyribonucleotide or ribonucleotide bases read from the 5′to the 3′ end. Polynucleotides include RNA and DNA, and may be isolatedfrom natural sources, synthesized in vitro, or prepared from acombination of natural and synthetic molecules.

[0027] The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

[0028] The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

[0029] The term “promoter” denotes a portion of a gene containing DNAsequences that provide for the binding of RNA polymerase and initiationof transcription. Promoter sequences are commonly, but not always, foundin the 5′ non-coding regions of genes.

[0030] The term “secretory signal sequence” denotes a DNA sequence thatencodes a polypeptide (a “secretory peptide”) that, as a component of alarger polypeptide, directs the larger polypeptide through a secretorypathway of a cell in which it is synthesized. The larger peptide iscommonly cleaved to remove the secretory peptide during transit throughthe secretory pathway.

[0031] The term “receptor” denotes a cell-associated protein that bindsto a bioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

[0032] The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

[0033] A “soluble protein” is a protein polypeptide that is not bound toa cell membrane.

[0034] Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of the DNA ofSEQ ID NO:1, or a sequence complementary thereto, under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is about0.02 M or less at pH 7 and the temperature is at least about 60° C. Aspreviously noted, the isolated polynucleotides of the present inventioninclude DNA and RNA. Methods for isolating DNA and RNA are well known inthe art. Total RNA can be prepared using guanidine HCl extractionfollowed by isolation by centrifugation in a CsCl gradient [Chirgwin etal., Biochemistry 18:52-94 (1979)]. Poly (A)⁺ RNA is prepared from totalRNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA69:1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. Polynucleotides encoding Zcyto7 polypeptidesare then identified and isolated by, for example, hybridization or PCR.

[0035] Additionally, the polynucleotides of the present invention can besynthesized using a DNA synthesizer. Currently the method of choice isthe phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. For the production of longer genes (>300 bp),however, special strategies must be invoked, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. In addition to the protein codingsequence, synthetic genes can be designed with terminal sequences thatfacilitate insertion into a restriction endonuclease sites of a cloningvector and other sequences should also be added that contain signals forthe proper initiation and termination of transcription and translation.

[0036] See Glick, Bernard R. and Jack J. Pasternak, MolecularBiotechnology, Principles & Applications of Recombinant DNA,(ASM Press,Washington, D.C. 1994), Itakura, K. et al. Synthesis and use ofsynthetic oligonucleotides. Annu. Rev. Biochem. 53: 323-356 (1984), andClimie, S. et al. Chemical synthesis of the thymidylate synthase gene.Proc. Natl. Acad. Sci. USA 87:633-637 (1990).

[0037] Those skilled in the art will recognize that the sequencesdisclosed in SEQ ID NOS:1 and 2 represent a single allele of the human.There are a number of naturally occurring mature N-terminal variantshaving the leader sequence cleaved at differing positions. They includethe sequences defined by SEQ ID NOs 14, 36, 37 and 38. Allelic variantsof these sequences can be cloned by probing cDNA or genomic librariesfrom different individuals according to standard procedures. Examples ofvariants of human Zcyto7 are represented by the polypeptides of SEQ IDNOs: 15-25.

[0038] The murine Zcyto7 cDNA and protein are disclosed by SEQ ID NOs:11 and 12. The mature Zcyto7 polypeptide is defined by SEQ ID NOs: 39and 40.

[0039] The present invention further provides counterpart proteins andpolynucleotides from other species (“species orthologs”). Of particularinterest are Zcyto7 polypeptides from other mammalian species, includingmurine, porcine, ovine, bovine, canine, feline, equine, and otherprimates. Species orthologs of the human Zcyto7 protein can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses the protein. Suitable sources of mRNA can be identified byprobing Northern blots with probes designed from the sequences disclosedherein. A library is then prepared from mRNA of a positive tissue orcell line. A protein-encoding cDNA can then be isolated by a variety ofmethods, such as by probing with a complete or partial human or mousecDNA or with one or more sets of degenerate probes based on thedisclosed sequences. A cDNA can also be cloned using the polymerasechain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the sequences disclosed herein. Within an additionalmethod, the cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to the protein. Similar techniques can also be applied to theisolation of genomic clones. As used and claimed the language “anisolated polynucleotide which encodes a polypeptide, said polynucleotidebeing defined by SEQ ID NO: 2” includes all allelic variants and speciesorthologs of the polypeptide of SEQ ID NO:2.

[0040] The present invention also provides isolated protein polypeptidesthat are substantially homologous to the protein polypeptides of SEQ IDNO: 2 and its species orthologs. By “isolated” is meant a protein orpolypeptide that is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated polypeptide is substantially free of otherpolypeptides, particularly other polypeptides of animal origin. It ispreferred to provide the polypeptides in a highly purified form, i.e.greater than 95% pure, more preferably greater than 99% pure. The term“substantially homologous” is used herein to denote polypeptides having50%, preferably 60%, more preferably at least 80%, sequence identity tothe sequence shown in SEQ ID NO:2,or its species orthologs. Suchpolypeptides will more preferably be at least 90% identical, and mostpreferably 95% or more identical to SEQ ID NO:2,or its speciesorthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616(1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919 (1992). Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “blossom 62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 2 (amino acids are indicated bythe standard one-letter codes). The percent identity is then calculatedas:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}}  \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\{{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}} \\ {sequences} \rbrack\end{matrix}} \times 100$

TABLE 2 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

[0041] Sequence identity of polynucleotide molecules is determined bysimilar methods using a ratio as disclosed above.

[0042] Substantially homologous proteins and polypeptides arecharacterized as having one or more amino acid substitutions, deletionsor additions. These changes are preferably of a minor nature, that isconservative amino acid substitutions (see Table 3) and othersubstitutions that do not significantly affect the folding or activityof the protein or polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or a small extension that facilitatespurification (an affinity tag), such as a poly-histidine tract, proteinA [Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al., MethodsEnzymol. 198:3, (1991)], glutathione S transferase [Smith and Johnson,Gene 67:31, (1988)], or other antigenic epitope or binding domain. See,in general Ford et al., Protein Expression and Purification 2: 95-107(1991. DNAs encoding affinity tags are available from commercialsuppliers (e.g., Pharmacia Biotech, Piscataway, N.J.). TABLE 3Conservative amino acid substitutions Basic: arginine lysine histidineAcidic: glutamic acid aspartic acid Polar: glutamine asparagineHydrophobic: leucine isoleucine valine Aromatic: phenylalaninetryptophan tyrosine Small: glycine alanine serine threonine methionine

[0043] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis[Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al., Proc.Natl. Acad. Sci. USA 88:4498-4502 (1991)]. In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity (e.g., ligand binding and signal transduction) to identifyamino acid residues that are critical to the activity of the molecule.Sites of ligand-protein interaction can also be determined by analysisof crystal structure as determined by such techniques as nuclearmagnetic resonance, crystallography or photoaffinity labeling. See, forexample, de Vos et al., Science 255:306-312 (1992); Smith et al., J.Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64(1992). The identities of essential amino acids can also be inferredfrom analysis of homologies with related proteins.

[0044] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer, Science 241:53-57 (1988) or Bowie and Sauer,Proc. Natl. Acad. Sci. USA 86:2152-2156 (1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display, e.g., Lowman et al., Biochem. 30:10832-10837 (1991);Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis, Derbyshire et al., Gene46:145 (1986); Ner et al., DNA 7:127 (1988).

[0045] Mutagenesis methods as disclosed above can be combined withhigh-throughput screening methods to detect activity of cloned,mutagenized proteins in host cells. Preferred assays in this regardinclude cell proliferation assays and biosensor-based ligand-bindingassays, which are described below. Mutagenized DNA molecules that encodeactive proteins or portions thereof (e.g., ligand-binding fragments) canbe recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

[0046] Using the methods discussed above, one of ordinary skill in theart can prepare a variety of polypeptides that are substantiallyhomologous to SEQ ID NO:2 or allelic variants thereof and retain theproperties of the wild-type protein. As expressed and claimed herein thelanguage, “a polypeptide as defined by SEQ ID NO: 2” includes allallelic variants and species orthologs of the polypeptide.

[0047] Another embodiment of the present invention provides for apeptide or polypeptide comprising an epitope-bearing portion of apolypeptide of the invention. The epitope of the this polypeptideportion is an immunogenic or antigenic epitope of a polypeptide of theinvention. A region of a protein to which an antibody can bind isdefined as an “antigenic epitope”. See for instance, Geysen, H. M. etal., Proc. Natl. Acad Sci. USA 81:3998-4002 (1984).

[0048] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in the art that relativelyshort synthetic peptides that mimic part of a protein sequence areroutinely capable of eliciting an antiserum that reacts with thepartially mimicked protein. See Sutcliffe, J. G. et al. Science219:660-666 (1983). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Peptides that areextremely hydrophobic and those of six or fewer residues generally areineffective at inducing antibodies that bind to the mimicked protein;longer soluble peptides, especially those containing proline residues,usually are effective.

[0049] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.Antigenic epitope-bearing peptides and polypeptides of the presentinvention contain a sequence of at least nine, preferably between 15 toabout 30 amino acids contained within the amino acid sequence of apolypeptide of the invention. However, peptides or polypeptidescomprising a larger portion of an amino acid sequence of the invention,containing from 30 to 50 amino acids, or any length up to and includingthe entire amino acid sequence of a polypeptide of the invention, alsoare useful for inducing antibodies that react with the protein.Preferably, the amino acid sequence of the epitope-bearing peptide isselected to provide substantial solubility in aqueous solvents (i.e.,the sequence includes relatively hydrophilic residues and hydrophobicresidues are preferably avoided); and sequences containing prolineresidues are particularly preferred. All of the polypeptides shown inthe sequence listing contain antigenic epitopes to be used according tothe present invention, however, specifically designed antigenic epitopesinclude the peptides defined by SEQ ID NOs: 27-35.

[0050] Polynucleotides, generally a cDNA sequence, of the presentinvention encode the above-described polypeptides. A cDNA sequence whichencodes a polypeptide of the present invention is comprised of a seriesof codons, each amino acid residue of the polypeptide being encoded by acodon and each codon being comprised of three nucleotides. The aminoacid residues are encoded by their respective codons as follows.

[0051] Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;

[0052] Cysteine (Cys) is encoded by TGC or TGT;

[0053] Aspartic acid (Asp) is encoded by GAC or GAT;

[0054] Glutamic acid (Glu) is encoded by GAA or GAG;

[0055] Phenylalanine (Phe) is encoded by TTC or TTT;

[0056] Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;

[0057] Histidine (His) is encoded by CAC or CAT;

[0058] Isoleucine (Ile) is encoded by ATA, ATC or ATT;

[0059] Lysine (Lys) is encoded by AAA, or AAG;

[0060] Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;

[0061] Methionine (Met) is encoded by ATG;

[0062] Asparagine (Asn) is encoded by AAC or AAT;

[0063] Proline (Pro) is encoded by CCA, CCC, CCG or CCT;

[0064] Glutamine (Gln) is encoded by CAA or CAG;

[0065] Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;

[0066] Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;

[0067] Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;

[0068] Valine (Val) is encoded by GTA, GTC, GTG or GTT;

[0069] Tryptophan (Trp) is encoded by TGG; and

[0070] Tyrosine (Tyr) is encoded by TAC or TAT.

[0071] It is to be recognized that according to the present invention,when a cDNA is claimed as described above, it is understood that what isclaimed are both the sense strand, the anti-sense strand, and the DNA asdouble-stranded having both the sense and anti-sense strand annealedtogether by their respective hydrogen bonds. Also claimed is themessenger RNA (mRNA) which encodes the polypeptides of the presentinvention, and which mRNA is encoded by the above-described cDNA. Amessenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined above, with the exception that each thymine nucleotide (T)is replaced by a uracil nucleotide (U).

[0072] The protein polypeptides of the present invention, includingfull-length proteins, protein fragments (e.g. receptor-bindingfragments), and fusion polypeptides can be produced in geneticallyengineered host cells according to conventional techniques. Suitablehost cells are those cell types that can be transformed or transfectedwith exogenous DNA and grown in culture, and include bacteria, fungalcells, and cultured higher eukaryotic cells. Eukaryotic cells,particularly cultured cells of multicellular organisms, are preferred.Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, (2nd ed.) (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and Ausubel etal., ibid.

[0073] In general, a DNA sequence encoding a Zcyto7 polypeptide isoperably linked to other genetic elements required for its expression,generally including a transcription promoter and terminator, within anexpression vector. The vector will also commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0074] To direct a Zcyto7 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the protein, or may bederived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is joined to the Zcyto7 DNA sequencein the correct reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the polypeptide of interest,although certain signal sequences may be positioned elsewhere in the DNAsequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830).

[0075] Cultured mammalian cells are preferred hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection, Wigler et al.,Cell 14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603(1981): Graham and Van der Eb, Virology 52:456 (1973), electroporation,Neumann et al., EMBO J. 1:841-845 (1982), DEAE-dextran mediatedtransfection, Ausubel et al., eds., Current Protocols in MolecularBiology (John Wiley and Sons, Inc., NY, 1987), and liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone etal., Focus 15:80 (1993). The production of recombinant polypeptides incultured mammalian cells is disclosed, for example, by Levinson et al.,U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiteret al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650),COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No.CRL 10314), 293 [ATCC No. CRL 1573; Graham et al., J. Gen. Virol.36:59-72 (1977)] and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL61) cell lines. Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Rockville, Md. In general, strong transcription promotersare preferred, such as promoters from SV-40 or cytomegalovirus. See,e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include thosefrom metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) andthe adenovirus major late promoter.

[0076] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

[0077] Other higher eukaryotic cells can also be used as hosts,including insect cells, plant cells and avian cells. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No.4,775,624; and WIPO publication WO 94/06463. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58 (1987).

[0078] Fungal cells, including yeast cells, and particularly cells ofthe genus Saccharomyces, can also be used within the present invention,such as for producing protein fragments or polypeptide fusions. Methodsfor transforming yeast cells with exogenous DNA and producingrecombinant polypeptides therefrom are disclosed by, for example,Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No.4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No.5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cellsare selected by phenotype determined by the selectable marker, commonlydrug resistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use in yeast isthe POT1 vector system disclosed by Kawasaki et al., U.S. Pat. No.4,931,373, which allows transformed cells to be selected by growth inglucose-containing media. Suitable promoters and terminators for use inyeast include those from glycolytic enzyme genes (see, e.g., Kawasaki,U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; andBitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459-3465 (1986) andCregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

[0079] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

[0080] Within one aspect of the present invention, a novel protein isproduced by a cultured cell, and the cell is used to screen for areceptor or receptors for the protein, including the natural receptor,as well as agonists and antagonists of the natural ligand.

[0081] Protein Isolation:

[0082] Expressed recombinant polypeptides (or chimeric polypeptides) canbe purified using fractionation and/or conventional purification methodsand media. Ammonium sulfate precipitation and acid or chaotropeextraction may be used for fractionation of samples. Exemplarypurification steps may include hydroxyapatite, size exclusion, FPLC andreverse-phase high performance liquid chromatography. Suitable anionexchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carboduimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988).

[0083] The polypeptides of the present invention can be isolated byexploitation of their properties. For example, immobilized metal ionadsorption (IMAC) chromatography can be used to purify histidine-richproteins. Briefly, a gel is first charged with divalent metal ions toform a chelate [E. Sulkowski, Trends in Biochem. 3:1-7 (1985)].Histidine-rich proteins will be adsorbed to this matrix with differingaffinities, depending upon the metal ion used, and will be eluted bycompetitive elution, lowering the pH, or use of strong chelating agents.Other methods of purification include purification of glycosylatedproteins by lectin affinity chromatography and ion exchangechromatography [Methods in Enzymol., Vol. 182:529-39, “Guide to ProteinPurification”, M. Deutscher, (ed.), (Acad. Press, San Diego, 1990).Alternatively, a fusion of the polypeptide of interest and an affinitytag (e.g., polyhistidine, maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification. Furthermore, tofacilitate purification of the secreted receptor polypeptide, an aminoor carboxyl-terminal extension, such as a poly-histidine tag, substanceP, FLAG® peptide [Hopp et al., Bio/Technology 6:1204-1210 (1988);available from Eastman Kodak Co., New Haven, Conn.), a Glu-Glu affinitytag [Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985)],or another polypeptide or protein for which an antibody or otherspecific binding agent is available, can be fused to Zyto7 to aid inpurification.

[0084] Uses

[0085] Northern blot analysis of the expression of Zcyto7 reveals thatZcyto7 is specifically expressed in the spinal cord. In situ analysis ofthe spinal cord reveals that this expression is localized in the neuronsand dorsal root ganglia. Therefore, Zcyto7 may play a role in themaintenance of spinal cord involving either glial cells or neurons. Thisindicates that Zcyto7 can be used to treat a variety ofneurodegenerative diseases such as amyotrophic lateral sclerosis (ALS),or dymyelinating diseases including multiple sclerosis. Zcyto7 may alsobe used to treat sensory neuropathis. The tissue specificity of Zcyto7expression suggests that Zcyto7 may be a growth and/or maintenancefactor in the spinal cord.

[0086] Zcyto7 gene's location on chromosome 5 indicates that zcyto7 is acytokine which can be used to modulate the activities of cells of theimmune system. Zcyto7 can also be used as a chemoattractant ofneutrophils in the spinal column. This would be useful as ananti-infective for infections in the spinal column. It could also beused to help regulate other cytokines in the spinal cord. Zcyto7 mayalso be administered to treat peripheral neuropathies such asCharcot-Marie-Tooth (CMT) disease which is localized to the samechromosomal region of 5q as Zcyto7.

[0087] The fact that Zcyto7 inhibits the growth of BAF-3 and TF-1 cellsas shown in examples 11 and 12 below indicates that Zcyto7 can be usedto treat autoimmune diseases and possibly such cancers such asleukemias.

[0088] The present invention also provides reagents which will find usein diagnostic applications. For example, the Zcyto7 gene is heavilyexpressed in the spinal cord. A probe comprising the Zcyto7 DNA or RNAor a subsequence thereof can be used to determine if the Zcyto7 gene ispresent on chromosome 5 or if a mutation has occurred.

[0089] The present invention also provides reagents with significanttherapeutic value. The Zcyto7 polypeptide (naturally occurring orrecombinant), fragments thereof, antibodies and anti-idiotypicantibodies thereto, along with compounds identified as having bindingaffinity to the Zcyto7 polypeptide, should be useful in the treatment ofconditions associated with abnormal physiology or development, includingabnormal proliferation, e.g., cancerous conditions, or degenerativeconditions. For example, a disease or disorder associated with abnormalexpression or abnormal signaling by a Zcyto7 polypeptide should be alikely target for an agonist or antagonist of the Zcyto7 polypeptide.

[0090] In particular, Zcyto7 can be used to treat inflammation.Inflammation is a result of an immune response to an infection or as anautoimmune response to a self-antigen.

[0091] Treatment dosages should be titrated to optimize safety andefficacy. Methods for administration include intravenous, peritoneal,intramuscular, subdural, into the spinal fluid or transdermaladministration. Pharmaceutically acceptable carriers will include water,saline, buffers to name just a few. Dosage ranges would ordinarily beexpected from 0.1 μg to 1 mg per kilogram of body weight per day.Preferably, 1 μg to 100 μg per day. However, the doses by be higher orlower as can be determined by a medical doctor with ordinary skill inthe art. For a complete discussion of drug formulations and dosageranges see Remington's Pharmaceutical Sciences,17^(th) Ed., (MackPublishing Co., Easton, Pa., 1990), and Goodman and Gilman's: ThePharmacological Bases of Therapeutics, 9^(th) Ed. (Pergamon Press 1996).

[0092] Use of Zcyto7 to Promote Bone and Cartilage Growth

[0093] It has been discovered that Zcyto7 stimulates the proliferationof both chondrocytes and osteoblasts as is shown below in Examples 7 and9 respectively. In addition, Zcyto7 also stimulates the steady statelevel of glycosaminoglycan present in chondrocyte cultures as shown inExample 8. Thus Zcyto7 can be used to stimulate both bone and cartilagegrowth in a variety of different therapeutic settings.

[0094] Zcyto7 can be implanted in a mammalian body so that the zcyto7 isin contact with osteoblasts such that osteoblast proliferation occursand bone growth is stimulated. For example, zcyto7 can be placed in amatrix [with or without a bone morphogenic protein (BMP)]. The BMPinduces the migration of mesenchymal osteoblast precursors to the siteand further induces differentiation of the mesenchymal cells intoosteoblast. Zcyto7 will then stimulate the further proliferation of theosteoblasts. A suitable matrix is made up of particles of porousmaterials. The pores must be of a dimension to permit progenitor cellmigration and subsequent differentiation and proliferation. An idealparticle size should be in the range of 70-850 mm, preferably 150-420mm. The matrix containing the zcyto7 can be molded into a shapeencompassing a bone defect. Examples of matrix materials areparticulate, demineralized, guanidine extracted, species-specific bone.Other potentially useful matrix materials include collagen, homopolymersand copolymers of glycolic acid and lactic acid, hydroxyapatite,tricalcium phosphate and other calcium phosphates. Zcyto7 can be appliedinto a matrix at a sufficient concentration to promote the proliferationof osteoblasts, preferably at a concentration of at least 1 μg/ml ofmatrix. A solution of zcyto7 can also be injected directly into the siteof a bone fracture or defect including areas of bone degeneration toexpedite healing of the fracture or defect site. Examples of BMPs andthe use of matrices to produce are disclosed in PCT applicationpublication number WO 92/07073, publication No. WO 91/05802,U.S. Pat.No. 5,645,591 and U.S. Pat. No. 5,108,753.

[0095] Zcyto7 can be further used to treat osteoporosis by administeringa therapeutically effective amount of zcyto7 to an individual. Apreferred dosage would be 1 μg of zcyto7 per kilogram of body weight perday.

[0096] As stated above, it has also been determined that zcyto7 can beused to promote the production of cartilage through its ability tostimulate the proliferation of chondrocytes. Zcyto7 can be injecteddirectly into the site where cartilage is to be grown. For example,zcyto7 can be injected directly in joints which have been afflicted withosteoarthritis or other injured joints in which the cartilage has beenworn down. An example of a case in which additional cartilage needs tobe grown is shoulders and knees of injured athletes.

[0097] Cartilage can also be grown by first removing chondrocytes froman individual, culturing the chondrocytes with zcyto7 so that theyproliferate and reimplanting the chondrocytes back into the individualwhere cartilage needs to be produced.

[0098] Zcyto7 can also be used to stimulate the regeneration of dentinor bone which has been lost due to periodontal disease. To do this, thesurrounding tissue should be thoroughly cleaned and a solution of Zcto7be administered, preferably by injection, into the site in which dentinregeneration is desired.

[0099] Antibodies to the zcyto7 polypeptide can be purified and thenadministered to a patient. These reagents can be combined fortherapeutic use with additional active or inert ingredients, e.g., inpharmaceutically acceptable carriers or diluents along withphysiologically innocuous stabilizers and excipients. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies,binding fragments thereof or single-chain antibodies of the antibodiesincluding forms which are not complement binding.

[0100] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicationsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in vivo administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Methods for administration include oral, intravenous, peritoneal,intramuscular, or transdermal administration. Pharmaceuticallyacceptable carriers will include water, saline, buffers to name just afew. Dosage ranges would ordinarily be expected from 1 μg to 1000 μg perkilogram of body weight per day. However, the doses may be higher orlower as can be determined by a medical doctor with ordinary skill inthe art. For a complete discussion of drug formulations and dosageranges see Remington's Pharmaceutical Sciences, 17^(th) Ed., (MackPublishing Co., Easton, Pa., 1990), and Goodman and Gilman's: ThePharmacological Bases of Therapeutics,9^(th) Ed. (Pergamon Press 1996).

[0101] Nucleic Acid-Based Therapeutic Treatment

[0102] If a mammal has a mutated or lacks a Zcyto7 gene, the Zcyto7 genecan be introduced into the cells of the mammal. In one embodiment, agene encoding a Zcyto7 polypeptide is introduced in vivo in a viralvector. Such vectors include an attenuated or defective DNA virus, suchas but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. A defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Examples of particular vectorsinclude, but are not limited to, a defective herpes virus 1 (HSV1)vector [Kaplitt et al., Molec. Cell. Neurosci.,2:320-330 (1991)], anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al., J. Clin. Invest., 90:626-630 (1992), and adefective adeno-associated virus vector [Samulski et al., J. Virol.,61:3096-3101 (1987); Samulski et al. J. Virol., 63:3822-3828 (1989)].

[0103] In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., Cell, 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.,62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Blood, 82:845 (1993).

[0104] Alternatively, the vector can be introduced by lipofection invivo using liposomes. Synthetic cationic lipids can be used to prepareliposomes for in vivo transfection of a gene encoding a marker [Felgneret al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987); see Mackey etal., Proc. Natl. Acad. Sci. USA, 85:8027-8031 (1988)]. The use oflipofection to introduce exogenous genes into specific organs in vivohas certain practical advantages. Molecular targeting of liposomes tospecific cells represents one area of benefit. It is clear thatdirecting transfection to particular cells represents one area ofbenefit. It is clear that directing transfection to particular celltypes would be particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides, e.g., hormones or neurotransmitters, andproteins such as antibodies, or non-peptide molecules could be coupledto liposomes chemically.

[0105] It is possible to remove the cells from the body and introducethe vector as a naked DNA plasmid and then re-implant the transformedcells into the body. Naked DNA vector for gene therapy can be introducedinto the desired host cells by methods known in the art, e.g.,transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter [see, e.g., Wu et al., J. Biol.Chem., 267:963-967 (1992); Wu et al., J. Biol. Chem., 263:14621-14624(1988)].

[0106] Zcyto7 polypeptides can also be used to prepare antibodies thatspecifically bind to Zcyto7 polypeptides. These antibodies can then beused to manufacture anti-idiotypic antibodies. As used herein, the term“antibodies” includes polyclonal antibodies, monoclonal antibodies,antigen-binding fragments thereof such as F(ab′)₂ and Fab fragments, andthe like, including genetically engineered antibodies. Antibodies aredefined to be specifically binding if they bind to a Zcyto7 polypeptidewith a K_(a) of greater than or equal to 10⁷/M. The affinity of amonoclonal antibody can be readily determined by one of ordinary skillin the art (see, for example, Scatchard, ibid.).

[0107] Methods for preparing polyclonal and monoclonal antibodies arewell known in the art (see for example, Sambrook et al., MolecularCloning: A Laboratory Manual, (Second Edition) (Cold Spring Harbor,N.Y., 1989); and Hurrell, J. G. R., Ed., Monoclonal HybridomaAntibodies: Techniques and Applications (CRC Press, Inc., Boca Raton,Fla., 1982). As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated from a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats. The immunogenicity of a Zcyto7 polypeptide may beincreased through the use of an adjuvant such as Freund's complete orincomplete adjuvant. A variety of assays known to those skilled in theart can be utilized to detect antibodies which specifically bind toZcyto7 polypeptides. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), (Cold SpringHarbor Laboratory Press, 1988). Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radio-immunoassays,radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA),dot blot assays, inhibition or competition assays, and sandwich assays.

[0108] As would be evident to one of ordinary skill in the art,polyclonal antibodies can be generated by inoculating a variety ofwarm-blooded animals such as horses, cows, goats, sheep, dogs, chickens,rabbits, mice, hamsters, guinea pigs and rats with a Zcyto7 polypeptideor a fragment thereof. The immunogenicity of a Zcyto7 polypeptide may beincreased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of Zcyto7 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like”, such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

[0109] As used herein, the term “antibodies” includes polyclonalantibodies, affinity-purified polyclonal antibodies, monoclonalantibodies, and antigen-binding fragments, such as F(ab′)₂ and Fabproteolytic fragments. Genetically engineered intact antibodies orfragments, such as chimeric antibodies, Fv fragments, single chainantibodies and the like, as well as synthetic antigen-binding peptidesand polypeptides, are also included. Non-human antibodies may behumanized by grafting non-human CDRs onto human framework and constantregions, or by incorporating the entire non-human variable domains(optionally “cloaking” them with a human-like surface by replacement ofexposed residues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

[0110] Alternative techniques for generating or selecting antibodiesuseful herein include in vitro exposure of lymphocytes to Zcyto7 proteinor peptide, and selection of antibody display libraries in phage orsimilar vectors (for instance, through use of immobilized or labeledZcyto7 protein or peptide). Genes encoding polypeptides having potentialZcyto7 polypeptide binding domains can be obtained by screening randompeptide libraries displayed on phage (phage display) or on bacteria,such as E. coli. Nucleotide sequences encoding the polypeptides can beobtained in a number of ways, such as through random mutagenesis andrandom polynucleotide synthesis. These random peptide display librariescan be used to screen for peptides which interact with a known targetwhich can be a protein or polypeptide, such as a ligand or receptor, abiological or synthetic macromolecule, or organic or inorganicsubstances. Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the Zcyto7sequences disclosed herein to identify proteins which bind to Zcyto7.These “binding proteins” which interact with Zcyto7 polypeptides can beused for tagging cells; for isolating homolog polypeptides by affinitypurification; they can be directly or indirectly conjugated to drugs,toxins, radionuclides and the like. These binding proteins can also beused in analytical methods such as for screening expression librariesand neutralizing activity. The binding proteins can also be used fordiagnostic assays for determining circulating levels of polypeptides;for detecting or quantitating soluble polypeptides as marker ofunderlying pathology or disease. These binding proteins can also act asZcyto7 “antagonists” to block Zcyto7 binding and signal transduction invitro and in vivo.

[0111] Antibodies can also be generated gene therapy. The animal isadministered the DNA or RNA which encodes Zcyto7 or an immunogenicfragment thereof so that cells of the animals are transfected with thenucleic acid and express the protein which in turn elicits animmunogenic response. Antibodies which then are produced by the animalare isolated in the form of polyclonal or monoclonal antibodies.

[0112] Antibodies to Zcyto7 may be used for tagging cells that expressthe protein, for affinity purification, within diagnostic assays fordetermining circulating levels of soluble protein polypeptides, and asantagonists to block ligand binding and signal transduction in vitro andin vivo.

[0113] Radiation hybrid mapping is a somatic cell genetic techniquedeveloped for constructing high-resolution, contiguous maps of mammalianchromosomes [Cox et al., Science 250:245-250 (1990)]. Partial or fullknowledge of a gene's sequence allows the designing of PCR primerssuitable for use with chromosomal radiation hybrid mapping panels.Commercially available radiation hybrid mapping panels which cover theentire human genome, such as the Stanford G3 RH Panel and the GeneBridge4 RH Panel (Research Genetics, Inc., Huntsville, Ala.), are available.These panels enable rapid, PCR based, chromosomal localizations andordering of genes, sequence-tagged sites (STSs), and othernonpolymorphic- and polymorphic markers within a region of interest.This includes establishing directly proportional physical distancesbetween newly discovered genes of interest and previously mappedmarkers. The precise knowledge of a gene's position can be useful in anumber of ways including: 1) determining if a sequence is part of anexisting contig and obtaining additional surrounding genetic sequencesin various forms such as YAC-, BAC- or cDNA clones, 2) providing apossible candidate gene for an inheritable disease which shows linkageto the same chromosomal region, and 3) for cross-referencing modelorganisms such as mouse which may be beneficial in helping to determinewhat function a particular gene might have.

[0114] The present invention also provides reagents which will find usein diagnostic applications. For example, the Zcyto7 gene has been mappedon chromosome 5q31. A Zcyto7 nucleic acid probe could to used to checkfor abnormalities on chromosome 5. For example, a probe comprisingZcyto7 DNA or RNA or a subsequence thereof can be used to determine ifthe Zcyto7 gene is present on chromosome 5q31 or if a mutation hasoccurred. Detectable chromosomal aberrations at the Zcyto7 gene locusinclude but are not limited to aneuploidy, gene copy number changes,insertions, deletions, restriction site changes and rearrangements. Suchaberrations can be detected using polynucleotides of the presentinvention by employing molecular genetic techniques, such as restrictionfragment length polymorphism (RFLP) analysis, short tandem repeat (STR)analysis employing PCR techniques, and other genetic linkage analysistechniques known in the art [Sambrook et al., ibid.; Ausubel, et. al.,ibid.; Marian, A. J., Chest, 108: 255-265, (1995)].

[0115] Zcyto7 maps at the 5q31 region which is a “gene cluster” whichcontains a group of cytokines and cytokine receptors. The cytokinesclustered there include IL-3, IL-4, IL-5, IL-13, GM-CSF, and M-CSF. Thisresult authenticates zcyto7 as a cytokine.

[0116] The invention is further illustrated by the followingnon-limiting examples.

EXAMPLE 1 Cloning of Zcyto7

[0117] Zcyto7 was identified from expressed sequence tag defined (EST)582069 (SEQ ID NO: 3) by its homology to Interleukin-17. The EST 582069cDNA clone was obtained from a human fetal heart cDNA library from theIMAGE consortium, Lawrence Livermore National Laboratory through GenomeSystems, Inc. The cDNA was supplied as an agar stab containing E. colitransfected with the plasmid having the cDNA of interest. The plasmidcontaining the cDNA was streaked out on an LB 100 μg/ml ampicillin and100 μg/ml methicillin plate. The cDNA insert was sequenced. The insertwas determined to be 717 base pairs long with a 180 amino acid openreading frame and a putative 20 amino acid signal peptide.

EXAMPLE 2 Northern Blot Analysis

[0118] Human multiple tissue blots 1,2,3 (Clontech)were probed todetermine the tissue distribution of Zcyto7. A EcoRI/NotI fragmentcontaining the entire Zcyto7 coding region was generated from theEST582069 clone and used for the probe. A plasmid prep of EST582069 wasprepared from a 5 ml LB 100 μg/ml ampicillin overnight culture at 37°using the QIAprep Spin Miniprep Kit (Qiagen). 12 μl out of 100 μl weredigested with 5 μl of H buffer (Boehringer Mannheim), 12.5 units ofEcoRI (Gibco BRL) and 12.5 units Notl (New England Biolabs) in a 50 μlreaction at 37° C. for 2 hours. The digest was electrophoresed on a 0.7%TBE agarose gel and the fragment was cut out. To obtain additionalmaterial the digest was repeated under the same conditions as aboveexcept 24 μl of EST582069 was used in the second digest. The seconddigest was electrophoresed on a 0.7% TBE agarose gel and the fragmentwas cut out. The DNA was extracted from both gel slabs with a QIAquickGel Extraction Kit (Qiagen). 135 ng of this DNA was labeled with P³²using the Multiprime DNA Labeling System (Amersham) and unincorporatedradioactivity was removed with a NucTrap Probe Purification Column(Stratagene). Multiple tissue northerns and a human RNA master blot wereprehybridized 3 hours with 10 ml ExpressHyb Solution (Clontech)containing 1 mg salmon sperm DNA which was boiled 5 minutes and theniced 1 minute and added to 10 ml of ExpressHyb Solution, mixed and addedto blots. Hybridization was carried out overnight at 65° C. Initial washconditions were as follows: 2×SSC, 0.05% SDS RT for 40 minutes withseveral changes of solution then 0.1×SSC, 0.1% SDS at 50° C. for 40minutes, 1 solution change. Blots were than exposed to film a −80° C.for 5 hours. There was cross hybridization/background so blots werefurther washed at 55° C. then 65° C. with 0.1%×SSC, 0.1% SDS for 1 houreach. Spinal cord showed very high expression of Zcyto7 mRNA and tracheashowed weak expression of mRNA. The transcript size was approximately0.75 kb.

EXAMPLE 3 Chromosomal Assignment and Placement of Zcyto7

[0119] Zcyto7 was mapped to chromosome 5 using the commerciallyavailable “GeneBridge 4 Radiation Hybrid Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panelcontains PCRable DNAs from each of 93 radiation hybrid clones, plus twocontrol DNAs (the HFL donor and the A23 recipient). A publicly availableWWW server (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl)allows mapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map of the human genome) which was constructed with theGeneBridge 4 Radiation Hybrid Panel.

[0120] For the mapping of Zcyto7 with the “GeneBridge 4 RH Panel”, 20

1 reactions were set up in a PCRable 96-well microtiter plate(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2

1 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6

1 dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1

1 sense primer SEQ ID NO:4, 1

1 antisense primer SEQ ID NO:5, 2

1 “RediLoad” (Research Genetics, Inc., Huntsville, Ala.), 0.4

1 50× Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25ng of DNA from an individual hybrid clone or control and x

1 ddH₂O for a total volume of 20

1. The reactions were overlaid with an equal amount of mineral oil andsealed. The PCR cycler conditions were as follows: an initial 1 cycle 5minute denaturation at 95° C., 35 cycles of a 1 minute denaturation at95° C., 1 minute annealing at 52° C. and 1 minute extension at 72° C.,followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 3% NuSieve GTG agarosegel (FMC Bioproducts, Rockland, Me.).

[0121] The results showed that Zcyto7 maps 490.89 cR from the top of thehuman chromosome 5 linkage group on the WICGR radiation hybrid map.Relative to the centromere, its nearest proximal marker was D5S413 andits nearest distal maker was WI-5208. The use of surrounding markersposition Zcyto7 in the 5q31.3-q32 region on the integrated LDBchromosome 5 map (The Genetic Location Database, University ofSouthhampton, WWW server:http://cedar.genetics.soton.ac.uk/public_html/).

EXAMPLE 4 Construction of Zcyto7 Expression Vectors

[0122] Two Zcyto7 construction vectors were made in a FLAG amino acidsequence (SEQ ID NO: 10) was inserted onto the N-terminal or C-terminalends of the Zcyto7 polypeptide. For the construction in which the FLAGamino acid sequence was attached to the N-terminus of Zcyto7, a 473 bpZcyto7 PCR DNA fragment was generated with 1 μl of a

dilution of the EST582069 plasmid prep of Example 2 and 20 picomoles(pm) of primer SEQ ID NO: 6 and 20 pm primer SEQ ID NO: 7. The PCRreaction was incubated at 94° C. for 1 minute, and then run for 5 cycleseach individual cycle being comprised of 20 seconds at 94° C. and 2minutes at 64° C. This was followed by 22 cycles each cycle beingcomprised of 20 seconds at 94° C. and 2 minutes at 74° C. The reactionwas ended with an incubation for 10 minutes at 74° C. 50 μl of the PCRreaction mixture was digested with 30 units of BamH1 (BoehringerMannheim) and 120 units of Xho1 (Boehringer Mannheim) for 2 hours at 37°C. The digested reaction mixture was electrophoresed on a 1% TBE gel;the DNA band was excised with a razor blade and the DNA was extractedfrom the gel with the Qiaquick<<Gel Extraction Kit (Qiagen). The excisedDNA was subcloned into plasmid nfpzp9 which had been cut with Bam andXho. Nfpzp9 is a mammalian cell expression vector comprising anexpression cassette containing the mouse metallothionein-1 promoter, asequence encoding the tissue plasminogen activator (TPA) leader, thenthe FLAG peptide (SEQ ID NO:10), then multiple restriction sites. Thesewere followed by the human growth hormone terminator, an E. coli originof replication and a mammalian selectable marker expression unitcontaining the SV40 promoter, enhancer and origin of replication; adihydrofolate reductase gene (DHFR) and the SV40 terminator.

[0123] For the construction of the zcyto7 gene in which a C-terminusFLAG was inserted onto the C-terminus of the zcyto7 polypeptide, a 543bp zcyto7 PCR fragment was generated with 1 μl of

dilution of the EST582069 plasmid preparation described in Example 1 and20 pm each of primers SEQ ID NO: 8 and SEQ ID NO: 9. The PCR reactionwas incubated at 94° C. for 1 minute, then run for 5 cycles, each cyclebeing comprised 20 seconds at 94° C. and 2 minutes at 55° C. This wasfollowed by 22 cycles each cycle comprised of 20 seconds at 94° C. and 2minutes at 74° C. The reaction was ended with a final 10 minuteextension at 74° C. The entire reaction mixture was run on a 1% TBE geland the DNA was cut out with a razor blade and the DNA was extractedusing the QIAQUICK™ gel extraction kit. 20 μl out of the recovered 35 μldigested with 10 units of BamH1 (Boehringer Mannheim) and 10 units ofEcoR1(Gibco BRL) for 2 hours at 37° C. The digested PCR mixture waselectrophoresed on a 1% TBE gel. The DNA band was cut out with a razorblade and the DNA was extracted from the gel using the QIAquick<<GelExtraction Kit (Qiagen). The extracted DNA was subcloned into plasmidcfpzp9 which had been cut with EcoR1 and BamH1. Plasmid cfpzp9 is amammalian expression vector containing an expression cassette having themouse metallothionein-1 promoter, multiple restriction sites forinsertion of coding sequences, a sequence encoding the FLAG peptide, SEQID NO:10, a stop codon, a human growth hormone terminator, an E. coliorigin of replication, a mammalian selectable marker expression unithaving an SV40 promoter, enhancer and origin of replication, a DHFR geneand the SV40 terminator.

[0124] Using antibodies to the FLAG polypeptides, one can separate theFLAG-tagged Zcyto7 polypeptides from a cell supernatant liquid.

EXAMPLE 5 Cloning of Murine Zcyto7

[0125] Mouse Zcyto7 was identified from EST 660242 SEQ ID NO:14 by itshomology to human Zcyto7. The cDNA clone was obtained from LawrenceLivermore National Laboratory through Genome Systems from a murineembryo cDNA library in which the embryos were between 13.5 and 14.5 daysold. The cDNA was supplied as an agar stab containing E. colitransfected with the plasmid having the cDNA of interest and thenstreaked out on an LB 100 μg/ml ampicillin, 25 μg/ml methicillin plate.The cDNA insert in EST660242 was sequenced. The insert was determined tobe 785 base pairs with an open reading frame of 180 amino acids and aputative 20 amino acid signal peptide. The sequences are defined by SEQID NO:11 and SEQ ID NO:12.

EXAMPLE 6 Tissue Distribution of Murine Zcyto7

[0126] Mouse Multiple Tissue Northern Blot (Clontech, Palo Alto,Calif.), mouse northern dot blot (Clontech), a mouse embryo northernblot, and a mouse spinal cord dot blot were probed to determine thetissue distribution of murine Zcyto7.

[0127] The mouse embryo RNA were isolated from mouse embryos which were6 to nine days from the date of fertilization using the POLY (A) PURE®mRNA isolation kit (Ambion). 100 mg of each mouse embryo was lysed in 1ml of lysis buffer, homogenized and processed in batch method accordingto the manufacturer's protocol. For the northern blot, 2 μg of RNA wasloaded on 1.5% agarose, 2.2M formaldehyde gel. The gel was run at 60Vfor four hours and 30 minutes. The RNA was transferred overnight onto aNytran membrane which had been pre-wetted in 20×SSC. The RNAs werecrosslinked onto the membrane by UV light and baked at 80° C. for 1hour.

[0128] The mouse spinal cord RNA was also prepared with the POLY (A)PURE® mRNA isolation kit (Ambion). The mouse spinal cord dot blot wasmade by spotting a dot with 1, 2 and 3 μl of RNA at 1 μg RNA/μlconcentration onto Nytran membrane.

[0129] A Not 1/EcoRI fragment containing the entire Zcyto7 coding regionwas generated from the clone containing SEQ ID NO: 12 (hereinafterreferred to as the SEQ ID NO:12 clone) and was used for the probe. Aplasmid prep of the SEQ ID NO:12 clone was prepared from a 5 ml LB 100μg/ml ampicillin overnight culture at 37° C. using the QIAPREP SPINMINIPREP kit (Qiagen). 4.66 μg were digested with 8 μl of high buffer(Boehringer Mannheim), 20 units of Not1 (Biolabs) and 20 units of EcoRI(Gibco BRL) in a 80 μl reaction at 37° C. for 2 hours. The digest waselectrophoresed on a 1.0% TBE gel and the fragment was cut out. The DNAwas extracted from the gel slab with a QIAQUICK® gel extraction kit(Qiagen). 98.8 ng of this fragment was labeled with P³² using theMULTIMPRIME® DNA labeling system (Amersham) and unincorporatedradioactivity was removed with a NUCTRAP® probe purification column(Stratagene).

[0130] The two northern preps and the two dot blot preps wereprehybridized for 3 hours at 65° C. as follows. 1 mg of salmon sperm wasboiled 5 minutes, iced 1 minute, mixed with 10 ml of EXPRESSHYB®solution and added to the blots. Hybridization was carried out overnightat 65° C. Initial wash conditions were as follows: 2×SSC, 0.1% SDS for40 minutes at room temperature then 0.1×SSC, 0.1% SDS for 40 minutes at50° C. Blots were exposed to film overnight at −80° C. The northernblots and the mouse dot blot were further washed with 0.1%×SSC, 0.1% SDSat 60° C. to remove background. The mouse spinal cord dot blot waswashed again at higher stringency with 0.1×SSC, 0.1% SDS at 65° C. toconfirm the earlier results.

[0131] Results: Mouse Zcyto7 expression was seen in the spinal cord,submaxillary gland and epididymis. Mouse embryo showed expression ofZcyto7 starting on day 12, peaking at day 16 and ending day 17 from thedate of fertilization. The transcript size was approximately 1 kb.

EXAMPLE 7 Proliferation of Chondrocytes Using Zcyto7

[0132] A chondrocyte proliferation assay was done to determine theeffect that Zcyto7 would have on chondrocyte proliferation. The assaywas done with 20% confluent cultures. As a control vehicle, bovine serumalbumin was added to a culture of chondrocytes instead of Zcyto7. Theassay measured the 3H-thymidine incorporation of nascent DNA in thechondrocytes, Wahl et al., Mol. Cell. Biol. 8:5016-5025 (1988).

[0133] Results: A 3.5—9 fold stimulation of primary chondrocyteproliferation was seen upon exposure of the chondrocyte cultures to 1μg/ml of zcyto7. Chondrocyte stimulation by zcyto7 was seen withmultiple preparations of the protein and was seen across species lines.It contrast to this, the control experiment using BSA resulted in nostimulation of chondrocytes.

EXAMPLE 8 Production of Glycosaminoglycan by Zcyto7-treated Chondrocytes

[0134] A 20% confluent culture of chondrocytes was prepared and zcyto7was applied at a concentration of 1 μg/ml. In a second experiment inaddition to zcyto7, IL-1β was applied to the cell culture. In a controlgroup BSA was added to the chondrocyte culture. The level ofglycosaminoglycan (GAG) production by the chondrocyte culture was thendetermined using a 1,9-dimethylmethlyene blue dye binding assay, Fardaleet al., Biochem. Biophys. Acta 888:173-177 (1987).

[0135] Results: Chondrocytes which were cultured with zcyto7 showed a50% increase in the steady state presence of GAG in the chondrocyteculture. Moreover, when the chondrocytes were co-cultured with bothzcyto7 and interleukin-1β (IL-1)the GAG production by the chondrocytesincreased 2.5 fold as compared with culturing of the chondrocytes witheither zcyto7 or EL-1β alone. While the cultured cells to which BSA wasadded showed no increased production of GAG.

EXAMPLE 9 Osteoblast Stimulation by Zcyto7

[0136] The CCC4 cell line is an osteoblast-like cell line derived fromp53 knockout mice. The CCC4 line was transfected with a plasmidcontaining an inducible serum response element (SRE) driving theexpression of luciferase. The stimulation of the SRE and thus theexpression of luciferase indicates that the chemical entity is likely tostimulate osteoblasts.

[0137] CCC4 cells were cultured in the presence of 1 μg of zcyto7/ml ofculture medium. As a control BSA, fibroblast growth factor (FGF) andplatelet-derived growth factor (PDGF) were each added to differentcultures of CCC4 cells. BSA was a negative control and FGF and PDGF werepositive controls as they are known to promote osteoblast proliferation.Luciferase activity was detected by addition of 40 μl of Promegaluciferase substrate using a 2 second integrated read on LabsystesLUMINOSKAN^(<<).

[0138] Results

[0139] Zcyto7 as well as, FGF and PDGF stimulate the expression ofluciferase in this assay indicating that they stimulate osteoblasts. TheBSA vehicle control was negative in this assay.

EXAMPLE 10 Effect of Zcyto7 on the Growth of Fibroblasts

[0140] Confluent cultures of human dermal, lung, and fetal lungfibroblasts were inoculated with Zcyto7 to determine the effects ofZcyto7 on the growth of fibroblasts. FGF was used as a positive controland BSA as a negative control vehicle.

[0141] Results: Zcyto7 had no effect on the growth of fibroblasts.

EXAMPLE 11 Effect of Zcyto7 on the Growth of BaF3 Cells

[0142] BaF3 cells, a murine pre-B cell line dependent on IL-3 toproliferate, were washed several times with base medium and then platedin a 96-well plate each well contained approximately 5500 cells/well.The cells were treated with either 1 μg/ml of Zcyto7 or 1-2 pg/ml ofIL-3 or with a combination of both Zcyto7 and IL-3. Also in a separateexperiment 0.1-10 ng/ml of TGFβ was added to the wells instead ofZcyto7. After incubation of the assay plate at 37° C. and 5% CO2 for 3-6days, 20 μl of ALAMAR blue was added to each well and the plate isincubated at 37° C. for 15-24 hours. The plate was then read with afluorometer with excitation wavelength of 544 m and emission wavelengthof 590 m. The assay was also scored by eye for stimulation or inhibitionof cell proliferation prior to the addition of the ALAMAR blue. The basemedium contained RPMI 1640+10% HIA-FBS+L-glutamine+Na pyruvate.

[0143] Results: Zcyto7 and TGFβ significantly inhibited the IL-3 drivenproliferation of BaF3 cells. However, when neutralizing antibodies toTGFβ were added in along with the Zcyto7, the Zcyto7 inhibition of theproliferation of the BaF3 cells was eliminated.

EXAMPLE 12 Effect of Zcyto7 on the Growth of TF-1 Cells

[0144] TF-1 cells, a human leukemia cell line which is GM-CSF or IL-1βdependent, were washed several times with base medium and then plated ina 96-well plate each well containing approximately 7000 cells/well. Thecells were co-cultured with 1 μg/ml of Zcyto7 and 100-200 μg/ml ofIL-1β. Also in a separate experiment TGFβ was added to the wells insteadof Zcyto7. After incubation of the assay plate at 37° C. and 5% CO2 for3-6 days, 20 μl of ALAMAR blue was added to each well and the plate isincubated at 37° C. for 15-24 hours. The plate is then read with afluorometer with excitation wavelength of 544 nm and emission wavelengthof 590 nm. The assay was also scored by eye for stimulation orinhibition of cell proliferation prior to the addition of the ALAMARblue. The base medium contained RPMI 1640+10% HIA-FBS+L-glutamine+Napyruvate.

[0145] Results: The IL-1β stimulation of TF-1 cells is inhibited by bothZcyto7 and TGF-β. The concentration of Zcyto7 at which inhibition ofproliferation occurred was greater than 200 ng/ml; and the concentrationof TGF-β at which inhibition of proliferation occurred was about 50pg/ml.

1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO:12 or the amino acid sequence of SEQ ID NO:39.